Process for producing the antibiotic U-44,590

ABSTRACT

New antibiotic U-44,590 and derivatives thereof, produced by the controlled fermentation of the new microorganism Streptomyces platensis var. clarensis var nova, NRRL 8035. This antibiotic and its derivatives are active against Gram-negative bacteria. Accordingly, they can be used in various environments to eradicate or control such bacteria.

United States Patent [1 1 DeBoer et al.

[ Sept. 23, 1975 PROCESS FOR PRODUCING THE ANTIBIOTIC U-44,590

[75] Inventors: Clarence DeBoer; Brian Bannister,

both of Kalamazoo, Mich.

[73] Assignee: The Upjohn Company, Kalamazoo,

Mich.

[22] Filed: Jan. 15, 1975 [21] Appl, No.: 541,346

Related US. Application Data [62] Division of Ser. No. 471,322, May 20, I974.

[52] US Cl 195/80 R [St] lnt. Cl.'- C12D 9/00 [58] Field of Search 195/80 R [56] References Cited UNITED STATES PATENTS 3.462,4l4 8/1969 Wechter 260/2l l.5

Primary E.\'aminerLionel M. Shapiro Assistant E.\'aminerRobert J. Warden Attorney, Agent, or FirmRoman Saliwanchik [57] ABSTRACT 3 Claims, N0 Drawings CROSS REFERENCE TO RELATED APPLICATION This application is a division of our copending application Ser. No. 471.322. filed on May 20. 1974.

BRIEF SUMMARY OF THE INVENTION The novel antibiotic of the invention. Ll-44.590 is obtained by culturing .S'Irepmnrvees' platens-is var. clurenxis. NRRI. 8035. in an aqueous nutrient medium under aerobic conditions. Various derivatives of 1344.590 can be made as disclosed infra. U44.590and its derivatives have the property of adversely affecting the growth of (iram-ncgative and Gram-positive bacteria. for example. Sire mu'ueeus /I(I)Zl)l \li('ll.. Klehsiella pneumonia. Salmonella Sp.. Serratia marcescens. Pastcuralla multocida. Hemop/zilux $11.. Proteus morgani and Proteus retlgeri. According. U-44.590 and its derivatives can be used along or in. combination with other antibiotic agents to prevent the. growth of or reduce the number of bacteria. as disclosed above. in various environments.

U-44.590 and its derivatives are also active against DNA viruses. for example. the Herpes virus and. thus. can be used to control such virus where its presence is not desired.

DETAILED DESCRIPTION OF THE INVENTION Chemical and Physical Properties of U-44.590

Elemental Analysis: Calcd. for C H N Q-p C. 44.08; H. 6.17; N. 17.13. Found: C. 44.14; H. 6.08; N. 1736.

Molecular Weight: 245 (Determined by mass spectrometry Melting Point Range: 141 142 C. v

Specific Rotation: [04],, =5 (c. 0.9030 in H O) Solubilities: Highly soluble in water. and lower alcohols. for example. methanol and ethanol; relatively insoluble in Me- CO. EtOAe. hydrocarbons. CHQCI: and CHCI lnlraredAbsorption Spectra: U-44.590 has a characteristic infrared absorption spectrum when suspended in a mineral oil mull. Peaks are observed at the following wave lengths expressed in reciprocal centimeters:

liand Frequency (Wznc Numbers) Intensity 3440 3-140 .140 3340 3 l)() RUM) RUIN) 2900 (N T Nttjol) 2030 (N) 2x00 (NI 1095 slt.

. 1083 1510 1503 1483 1463 IN) 1440 1411 1407 1390 1375 (N) 1350 1315 I300 1280 I270 -Continued Band l-requency (Wave Numbers) Intensity 1102 1243 I sh. l 195 l 165 l 133 H193 [(135 HIM) Hill 9145 971 043 X35 X72 szzsssszsz szsssz s Note. sh means a shoulder hand.

Band Frequency (Wave Numbers) Intensity 3440 3200 3080 3000 1970 2960 2935 2920 2870 1097 sh. 1685 1510 14142 14M 1437 I420 I406 I396 134) 1310 1298 I290 I275 sh. 1263 1243 1195 l l 133 Ill)? 1K5 I060 Hill) 985 971 942 X83 X71) H47 827 7"l 754 733 zsssszsszzzzsss zzsssszzzz zz sss'ssszzw Note: sh mean a shoulder hand.

Infrared band intensities, throughout this disclosure, are indicated as S. M. and W respectively and are up proximated in terms of the backgrounds in the vicinity ofthe bands. An S band is of the same order of intensity as the strongest in the spectrum; M bands are between A; and A; as intense as the strongest band; and. W bands are less than A as intense as the strongest band. These estimates are made on the basis of a percent transmission scale. 7 1

The following is considered to be the structure of LI- 44.590:

Thus, U-44.590 can be referred to by the trivial name 5.6-dihydro--azathymidinc. or by its chemical name I-( 2-deoxy-,B-D-erythro-pentofuranosyl )-5.6-dihydro- 5-methyl-s-triazine-2.4( IH.3H )-dione.

Antibacterial Activity of U-44.590

The above antibacterial spectrum was obtained by a standard agar dilution test with the following media and conditions:

Diffo Brain Heart Infusion Medium was used for all test bacteria except I. mulmcida andHenwp/zilus species which were grown in Difco Blooc Agar Base with 5% defibrinated rabbit blood. All were grown aerobically at 37 C. (except Hemophilus species. grown anaerobically) 16-18 hours. Inocula were grown overnite (l6l8 hours) at 37 C. and used to seed agar at the rate of 0.025 ml. of dilution (approximately 2500 to 25.000 bacteria per drop of inoculum).

In vivo testing of U-44.590 in mice infected with selected microorganisms is as follows:

Activity (CD in mg/kg) I52 (96-240) I00 (ho-I52) IOI (66-156) 62.5

Proteus mirabilis Proteus vulgaris -Continued Activity t(l).-.l. in mg/kg) Mice. u

Challenge Subcutaneous I ()ral Organism l.I) Route Route I50 Streptococcus hemolyticus I00 lt 0 ANTIVIRAL ACTIVITY OF U-44.590

The following is an example of the antiviral activity of antibiotieU-44.590. The antibiotic is administered subcutaneously to mice which are inoculated intravenously with Herpes simplex virus. Treatment is initiated two hours prior toviral infection and is followed by treatment four times daily for five consecutive days. A detailed account of the materialsand methods and results are as follows: I

Male mice. weightingapproximately 20 gm. each. are divided into 4 groups of 20. Group I is treated with saline. Group 2 with 400 mg./kg/dose (mkd) U-44.590. Group 3 with .200 mkd U-44.590. and Group 4 with 100 mkd U-44.590. The antibiotic is dissolved in saline and administered subcutaneously in the nape of the neck at 8 am. 12 noon. 4 p.m.. and 8 pm. on days 0, l. 2. 3. and 4. Herpes virus at l0""" dilution, 0.05 nil/mouse. equivalent to a viral dose of 40 LD s. is inoculated into the tail vein at 10 a.m. on day 0. Paralysis and death are recorder daily.

Hind leg paralysis usually preceded death by l-2 days. All mice died that became paralyzed. Death pattern of the 4 groups. as shown in the curves which follow. illustrates the dose response obtained. Statistical analysis of the results at day 11 indicates that all 3 treated groups are significantly different from the con.- trol group (I).

THE MICROORGANISM The microorganism used for the production of U- 44,S is .S'trepmmycar platensis var. clarensis. NRRL 8035. A permanent collection of the Northern Regional Research Laboratory. U.S. Department of Agriculture. Peoria. Illinois. USA.

The microorganism of this invention was studied and characterized by Alma Dietz of the Upjohn Research Laboratories. I

v A new soil isolate with hygroscopic spore masses. but with smooth. hat-shaped (crescent) or brazil-nutshaped (elliptical) spores. has been found to differ in certain characteristics from the type culture Streptomyces plalensis. An outstanding difference of the new culture, is the production of antibiotic U-44.590. The new isolate can be recognized as a variant of .S'trepmmyccs' platcnsix by its cultural. microscopic. and biochemical characteristics. Therefore. it is proposed that this new isolate be designated .S'trepmmyces platensix var; Clarenxis Dictz var. nova. Rule 7 of the International Code of Nomenclature of Bacteria [International Code of Nomenclature of Bacteria. 1966. Edited by the Editorial Board of the Judicial Commission of the International Committee on Nomenclature of Bacteria. Int. J. Syst. Bacteriol. 16: 459-490] was applied in designating the variety epithet.

, .S'rreplomyces pluunsis var. clarensis is compared with the type species Strepmmyces platens-is Pittenger and Antibiotic U- l l,590 vs. Herpes virus in Mice (2O mice perq ro gg).

i/kg ./close .-.----Q Saline ui i,590 e100 mg ./kg. /dose Days 2369. and two recently characterized strains: Slropmi myvvs plulensis NRRl. 3593 '[Evans. Ralph Henry .lr.. and Samuel Owen Thomas. 197 l Antibiotics AH272a and AH272B and process for producing same. U.S. Patent 3.592.925 l and .S'rrepmmvces platensis NRRL 3761 [Okuda. Tomohau, and Shigemi Awatagouchi. 1973. Antibiotics YL 704 and preparation thereof. U.S. Patent 3.718.742].

Color characteristics: Aerial growth white to yellow to gray. Moist black hygroscopic patches on some media. Melanin-negative. Appearance on Ektachrome [Diet2.

A. 1954. Ektachrome transparencies as aids in actino mycete classification. Ann. NY. Acad. Sci. 60:152-154] is given in Table 1. Reference color characteristics are given in Tables 2 and 3. The new culture may be placed in the White (W Yellow (Y and Gray (GY) color series of Tresner and Backus [Tresnen H.

D.. and E. J. Backus. i963. System of color wheels for strcptomyccte taxonomy. Appl. Microbiol. ll:335'-338|. Microscopic characteristics: Spore chains in tight spirals uncoiling to long open spirals. Spore chains spiral (S) in the sense of Pridham et al. [Pridham. T. G.. C. W. Hesseltine. and R. G. Benedict. 1958. A guide for the classification of streptomycctes according to selected groups. Placement of strains in morphological sections. Appl. Microbiol. 6:52-79]. Spore hat-shaped (crescent) or Brazil-nut (elliptical) shaped. Spores are the platenis-typc of Tresner and Backus [Tresner, H. D.. E. J. Backus, and Jean A. Hayes. 1967. Morphological spore types in the .S'treptamyces I1 \'gr0.sc0picu.v-likc complex. Appl. Microbiol. 15:637-639]. Spore silhouette smooth by direct observation with the electron microscope. Spore surface ridged with surface markings by the carbon replication technique of Dietz and Mathews [Dietz, A. and J. Mathews. 1962. Taxonomy by carbon replication. I. An examination of Strepmmyces hygroscopicux. Appl. Microbiol. 10:258-263]. Cultural and biochemical characteristics: See Table 4. infra.

Carbon utilization: (irowth on carbon compounds was determined in the synthetic medium of Pridham and Got'tlieb lPridhani, T. G.. and D. Gottlieb. 1948. The utilization of carbon compounds by some Actinomy cetales as an aid for species determination. J. Bacteriol. 56:107-1 14], Table 5 and in the synthetic medium of Shirling and Gottlieb lshirling. E. B. and D. Gottlieb. I966. Methods for characterization of Streptomyces species. Int. J. Syst. Bacteriol. 16:313-340], Tablev Temperature: The cultures grew well as l8- 37 C. on Bennett's. Czapeks sucrose. maltose-tryptone, and Hickey-Tresner agars. Optimum growth was at 24 37 C. The new culture and the type culture did not grow at 45 55 C. The cultures designated NRRL 3593 and NRRL 376i grew at 45 C. but not at 55C. Antibiotic-producing propcrtieszj See Table 7. infra. Source: Soil v V Type culture: Strpmmyces platensis Pittenger and Gottlieb NRRL 2364.

Type variety: .Strepmmycm platensis var. platensis NRRL 2364. Variety: Streptomyces platensis var. clarensis Dietz var. nova.

Table 1 Appearance ol Streptomyces platensis Cultures on Ektachrome*' Deter- mi- 7 g na- S. platensis var. S. platensis S. platensis Agar medium tion clarcnsis NRRL 8035 NRRL 2364 NRRL 3593 Bennett's Czapek"s sucrose Maltose-tryptone Peptone-iron t). l A Tyrosine Casein starch Lavender-gray with black patches Lavender-gray Pink-tan Lavender-gray 5 Surface R Reverse Pale gray-yellow Dietz. A. I954. liktachrome transparencies as aids in aetinomycete classification. Ann. Acad. Sci..60:l52'-l54 Table 2 Reference Color Characteristics of Streptomyees platensis Cultures De- Color Harmony Manual 3rd ed. 1948* termi- S. platensis na v. clarensis S. platensis S. platensis S. platensis Agar Medium tion NRRL 8035 NRRL 2364 NRRL 3593 NRRI. 376i Bennetts S 2ihtg) to dtg) Zl'etm) 2dctm) 2dc(ml R 2tb(g) Jgttg) IAgctg) lee-(g) P .1 Czapeks S a(g) to Zbatg) Tigetm) Zhatm) htni sucrose R Zlbtg) B'getg) I'Aictg) 2catg) p Maltose- S Ste-(g) Zgetm) to htm) Zdctm) Zdetm) tryptone R lVzl'hig) 12gctm) I'Agdgi Zgctm) P Hiekey- S Illitm) to Bl'ct m) 3ig( tn) 2dc(m) 3getm) Tresucr R Iec(g) to 2ie(g) Zeetm) llzgctg) lectm) p g Yeast extraet- S 3igtm) 3getm) Zdetm) to 2ha(m) 3ge(m) malt extract R lectg) 3ietg) l'klctg) Zgctm) uspg P i'lalgtm) Oatmeal S 3igtm) 3igtm) 3ge(m) Klitm) USP-3) R leetg) Jietg) Zgctgl Zcatg) Table 2 Continued Reference Color Characteristics of Streptomyees platensis Cultures l)e Color Harmony Manual 3rd ed. l948* termiplatensis v naelarensis S. platensis S. platensis S. platensis Agar Medium tion NRRL 8035 NRRL 2364 NRRL 3593 NRRL 376i lnorganie S 3li(|n) to 3gelm) 3li(m) v Zdetm) Zgetm) salts-starch R Zlhlg) Zealm) to 3ie(m) Zeetg) Zeetg) USP-4) l Zgct g) 3ie-trace (ilyeerol- S h( m) Jget m) 1 Zhat g) Zhat m) asparagine R Zeclg) Zgelm) Zlhtm) 3eatg) USP-5) P Jacobson. lii. I C. (iramille. and C. l3. Foss. [94%. Color harmony manual. 3rd ed. Container Corporation a! America. Chicago. Illinois "Kelly. l\'. l... and I). ll. Judd. I955. The ISCC'NBS method ofdesignating et iliirs and a dictionary ofeolor names. US. Dept. ('omm. (ire 553 S Surl'aee R Reverse l Pigment lg) gloss ehip surface (in) L matte chip surl'aee Table 3 Color Code for Table 2 Color Harmony Manual 3rd ed. 1948* NBS Circular 553, I955 Color Chip Color Name Color Chip Color Name a White 2o3gm White h Oyster white 263m White 264g Light gray lee Light eitron gray. putty lZlm Pale yellow-green l22g (irayish yellow-green l Valli Pastel yellow 87g Moderate yellow l /gge Dusty yellow lUZg Moderate greenish yellow lllSgm Grayish greenish yellow 1 Vziu Light antique gold 87gnt Moderate yellow l /zl. Gold I Vzlg Golden olive lll7g Moderate olive Zha Pearl. shell tint 92gm Yellowish white Zea Light ivory. eggshell 89gm Light yellow Zde Natural. string 93gm Yellowish gray Zee Biscuit. ecru. oatmeal. sand )Ogm (irayish yellow 2th Bamboo. hul'll straw. wheat 87g Moderate yellow 89m Pale yellow Zl'e 7 Silver gray 94g Light olive brown I l2gm Light olive gray Zgc Bamhoo, ehamois )(lgm (irayish yellow Zge (overt tan. griege )llgm (irayish yellow Zie Light mustard tan )lgm I Dark grayish yellow 94g Light olive brown lUog Light olive Zih Dark covert gray l l2m Light olive gray l 13g Olive gray Table 3 Continued Color Code for Table 2 Color Harmony Manual 3rd ed l948* NBS Circular 553. l)55** Color Chip Color Name Color Chip Color Name 3ca Pearl pink. shell 73gm Pale orange yellow 3l'e Silver gray o3gm Light hrownish gray 3gc Light tan 76gm Light yellowish brown 3ge Beige. camel 79m Light grayish yellowish brown 94m Light olive brown 3ie Camel. maple sugar. tan 76m Light yellowish brown 77g Moderate yellowish brown 3ig Beige brown mist brown 80m Grayish yellowish brown 95g Moderate olive brown 3li Beaver 80m Grayish yellowish hrown 95g Moderate olive brown Jacohson. l3. W. C. Granville. and C. F. Foss. I948. Color harmony manual. 3rd ed. Container Corporation of America. Chicago. Illinois Kelly. K. I... and I). ll. Judd. I955. The lSCC-NBS method of designating colors and a dictionary of color names. US.

Dept. (omnL Circt 553 Table 4 Cultural and Biochemical Characteristics ol .S'rrupmnzyt'ux plan-"six Cultures termi- S. plutcnsis nav. clarunsix S. plulenxix S. plulunxi- S. platens-ix Medium tion NRRL 8035 NRRL 2364 NRRL 3593 NRRL 376l Agar Peptone-iron Pale gray Pale gray Pale gray-pink Pale olivetan Tan Yellow-tan Yellow Yellow Melanin-negative Melanin-negative Melanin-negative Melanin-negative Calcium malate Pale gray Pale tan Pale gray Pale gray-white Gray Pale tan Gray Gray Malate not Malate not Malatc not Malate slightly soluhilized solubilized solubilized soluhili'lcd Glucose asparagine Trace pale gray Pale pink-tan Pale gray-pink Gray-white Pale olive-tan Pink-tan Pale yellow-tan Pale salmon Skim milk Tyrosine Xanthine Nutrient starch Yeast extractmalt extract Bennett's Czapeks sucrose Maltose-tryptone Hickey-Tresner (modified) Peptone-yeast extract-iron USP-6) w 11:: 21:12? wQ-uwm owwm 01mm 0171 wwww OOF O'UW 07540170 :n'a nth-aw Pale yellow Pale gray-pink Orange-tan Orange-tan Casein not soluhilized Pale gray-pink Yellow Yellow Tyrosine soluhilized Pale gray-pink Yellow Xanthine slightly soluhilized under growth Pale gray pink Yellow Starch hydrolyzed Gray with black patches Yellow-tan Yellow Lavender-gray with black patches Olive-gray Pale yellowolive Gray-white Yellow Pale yellow Lavcndergray Olive-green Olive-yellow Gray with black center Olive Pale gray Yellow Melanin-negative Pale pink on edge Pale orange Pale orange Casein not solubilized White Yellow Yellow Tyrosine soluhilized White Pale yellow Xanthine slightly soluhilized under growth Pale pink Yellow Starch hydrolyzed Pale pink-tan Red-tan Yellow Lavender-gray with black patches Red-tan Pink-tan Gray-black in center. light gray on edge Gray-green Pale gray Cream-yellowpink Pale yellow Black with gray edge Olive-tan Pale olive Pale yellow Melanin-negative Xanthine not soluhililed Pale gray-pink Lemon-yellow Yellow Starch hydrolyzed White Deep yellow Yellow Pale lavendergray Light olive Light t )live White Yellow Yellow Gray-white ()livc Pale olive Deep gray-white Light olive Light olive Trace white Yellow Melanin-negative Trace white on edge Yellow Yellow Casein not solubilized Pale gray-pink Orange-tan Orange-tan Tyrosine soluhilized Pale gray-pink Yellow Xanthine soluhilized under growth Pale gray-pink Pale yellow Starch hydrolyzed Pale gray-pink Yellow Heavy gray Cream-tan Sparse graywhite Cream Gray-white Pale olive-cream Pale tan Gray with black patches Pale olive-erea-n Yellow Melaninnegative Table 4 Continued Medium Cultural and Biochemical Characteristics of .S'irt'plmnyunrplulunxix Cultures (ielatin Plain Nutriettt Broth Synthetic nitrate Nutrient nitrate Litmus milk termination S. plult'mis t. t'lurt-nxis NRRL 8035 (iray with black patches Pale olive Melanin-negative White Yellow Trace liquefaction Trace vegetative growth Trace liquefaction Trace bottont growth Nitrate not reduced to nitrite Trace white surface ring l-'locculent bottom growth Nitrate not re dueed to nitrite surface pellicle Litmus reduced S. plur 'lixis N R Rl 2 3 64 Gray with black patches Pale cream-pink Melanin-negative Colorless surface pellicle and bottom growth Nitrate not reduced to nitrite Trace white surface ring Flocculent bottom growth Nitrate not re' cluced to nitrite Surface pellicle No change .81 plult'uxix NRRl. 3593 Pale gray-white Pale olive Melanin-negative Colorless vegetative growth Trace brown No liquefaction White Trace yellow No liquefaction Trace bottom growth Nitrate ttot reduced to nitrite Trace bottom growth Nitrate not reducetl to nitrite Surface pelliclc Partial peptoni S. pluluntrix NRRl. 37o] Pale salmon Pale red-tan Melaninnegativc Colorless vegetatire growth No liquefaction White No liquefaction Trace bottom growth Nitrate reduced to nitrite Trace bottom growth Nitrate reduced to nitrite Surface pellicle Partial peptoni- Partial peptoni pH 6.8 zation pH 7.0 zation pH (1.8 zation coagulation pH 7.0

S surface R reverse P pigment other characteristics Table 5 Utilization of Carbon Compounds by S!I'([)ll)lll \'('l'.\ pltiu'nxi'x Cultures in the Synthetic Medium of Pridham and (iottlieh* S. [)[dltlhfS v. clart'nsis S. [IIUICIN'LY S. plum/mix S. plum/mix NRRL 8035 NRRL 2364 NRRL 3593 NRRL 37m CONTROL (no carbon compound added) l. D-Xylose r 2. L-Arabinose t) l) 3. Rhumnose 4. D-Fructose l 5 D-Galztctose r y 4 (i. D-Glucose -l 7 D-Mannose i 8. Maltose -l; 9. Sucrose 10. Lactose -l ll. Cellobiose -l (f) 12. Raflinose 13. Dextrin l4. lnulin (l t-l l5. Soluble starch [6. (ilycerol [7 Dulcitol (rl-l l8. l)-Mannitol -t +1 I). D sorbitol -l 20. lnositol l II. Salicin (J 22. Phenol Z3. (.resol 24. Na Formate 25. Na Oxalate l) l) i 26. Na Turtrate (l 27. Na Salicylate 2'. Na Acetate ('l') (H t 29. Na Citrate 'l (+J ('l') 30. Na Suceinate H) I (l) Tableo Utilization of Carbon Compounds by .S'lrcp/uhrvav pluu-njvix Cultures S. plum: v. (Ill/'1'. .v S. pluli'nsis S. pluu'llxix S. [IlllltlUiA NRRl. 8035. NRRl. 2364 NRRl. 359,3 N RRl. 37m

CONTROLS i Negative-basal Slight growth Slight growth Slight growth Slight growth medium i j I Positive-basal Good growth (iood growth (iootl growth I (iood growth medium plus D-glucose CARBON COMPOUNDS L-Arahinose -l-+ 4+ :l' S ucrose H +t- +l- D-Xylose 1 lnositol -l-l- -l+ H H D-Mannitol H -H' D-Fructose v l+ -l-l Rhamnose i i Rafl'i nose -ll -l +-l 4 l Cellulose t 4 Strong utili/alion -l Positive utiliration l'lilization doubtful Utili/alion negative Sliirling. Ii. 8..

Table 7 and l). (iottlieb. who. Methods for charactcri/ation of .S'lrvplnnrvr-rzvspecies. Int. J 'Syst. liacteriol. Hell} 3411 The new compound of the invention is produced when the elaborating organism is grown in an aqueous nutrient medium under submerged aerobic conditions. It is to be understood. also. that for the preparation of limited amounts surface cultures and bottles can be employed. The organism is grown in a' nutrient medium containing a carbon source. for example. an assimilable carbohydrate. and a nitrogen source. for example. as assimilable nitrogen compound or proteinaceous material. Preferred carbon sources include glucose, brown sugar. sucrose. glycerol. starch. cornstarch. larctose. dextrin. molasses. and the like. Preferred nitrogen sources include cornsteep liquor. yeast. autolyzed brewers yeast with milk solids. soybean meal. cottonseed meal. cornmeal. milk solids. pancreatic digest of casein. tish meal. distillers solids. animal pcptone liquors. meat and bone scraps. and the like. Combinations of these carbon and nitrogen sources can be used advantageously. Trace metals. for example.. zinc. magnesium. manganese. cobolt. iron. and the like. need not be added to the fermentation media since tap water and unpurified ingredients are used as components of the medium prior to sterilization of the medium.

Production of the compound of the invention can be effected at any temperature conducive to satisfactory growth of the microorganism. for example. between about 18 and 40 C.. and preferably between about 20 and 32 C. Ordinarily. optimum production ofthe compound is obtained in about 5 to 15 days. The medium normally remains neutral during the fermentation. The final pH is dependent. in part, on the buffers present. if any and in part on the initial pH of the culture medium.

7 When growth is carried out in large vessels and tanks. it is preferable to use the vegetative form. rather than the spore form. of the microorganism for inoculation to avoid a pronounced lag in the production of the new compound and the attendant inefficient utilization of the equipment. Accordingly. it is desirable to produce a vegetative inoculum in a nutrient broth culture by inoculating this broth culture with an aliquot from a soil. liquid N agar plug. or a slant culture. When a young. active vegetative inoculum has thus been secured. it is transferred aseptically to large vessels or tanks. The medium in which the vegetative inoculum is produced can be the same as. or different from. that utilized for the production of the new compound. so long as a good growth of the microorganism is obtained.

A variety ofprocedures can be employed in the isolation and purification of the compound of the subject invention. for example. solvent extraction. partition chromatography. silica gel chromatography. liquidliquid distribution in at Craig apparatus. absorption on resins. and crystallization from solvents.

In a preferred recovery process the compound of the subject invention is recovered from the culture medium by separation of the mycelia and undissolved solids by conventional means. such as by filtration or centrifugation. The antibiotic is recovered from the filtered or centrifuged broth by adsorption on activated carbon. The activated carbon is then washed with water to remove some impurities. This is followed by clutions with acetone: water solutions which remove the antibiotic from the activated carbon. The acetone in the eluates is removed, advantageously by evaporation. and the remaining aqueous residue is lyophilized to afford a crude preparation of antibiotic U-44,590.

A preferred purification procedure is to subject a crude preparataion of U-44.590. as described above. to chromatography on silica gel from which U-44,59() is eluted. Fractions which show activity against the bacterium Klcbsiclla pncumoniac on a standard agar plate test, are pooled and taken to dryness to yield a relatively pure preparation of U-44.590. Further purification is achieved by acetylation to a crystalline diacetate derivative of U-44.59(). Zemplen lG. Zemplen and E. Pacsu. Ber., 62, 1613 l929)] de-csterifieation (transesterification) with sodium methoxidein methanol, and neutralization of the catalytic amount ofbas e with carbon dioxide gives the, free antibiot ic U- 4 4 590 which crystallizes-readily from methanol eithyl acetate togive a pure preparation of t .l-44,59(). v

Antibiotic U-44,590 isv active against Streptococcus lwmolylicus and, thus, can be used to disinfect instruments. utensils or surfaces when contaminated with this microorganism, where the inactivation of this microorganism is-dcsirable. Also,, U-44,590 is active against Escherichia coli and can be used to reduce, arrest, and/or: eradicate slime. production in papermill sys- 'tems because of its antibacterial aetionagainst this bacterium. Antibiotic U-44,590 can also .be used to prolong the life 'of cultures of-Trichomonasfoetus, Trichom'onas hminis,-'rand Trichomonas vaginalis by freeing them of Escherichia coli" contamination. Further, U-44,590 can be used to inhibitthe growth of E. coli in hospital flower vaseswhereithas-been reported to exist and present a hazard to hospital patients. See Clinical Medicine, February, 1974, Page 9.

Novel acyl derivatives of l- 44.590. disclosed" herein. can be used for-'the same antibiotic purposes as U=44.590 in environments possessing means todeacylate the compound to U,-44,590.Thus, the acyl dcrivatives of U-44.59() can be used to trca t laboratory mice infectcdwith a Gram-negative bacteria, fo 'rcxample 1:.

coli. asvdiseloscd herein. Further. acyl derivatives of U- EXAMPLE 1 Part A. Fermentation i A soil stock of S'lrcpmntvccs plu mmis v'ar. clurcnsis.

NRRL 8035 is used to inoculate a series of500-ml. Er-

lcnnicye'r flasks. each containing 100 ml. of sterile seed medium consisting of the following ingredients:

(ilucose monuhydratc l0 (rm/l Bacto Pcptonc IIJiIcol t [0 (int/l Bacto Yeast lixtract (Dilco) 2.5 (im/l Deionircd water Balance Brer Rabbit Molasses (RJR Foods lnc.. N.Y., NY. l00l7) 20ml Yeast Extract (Difco) Detroit.

Michigan l (jm/l (ilucose monohydrate ,1 l0 (im/l Dextrin (Corn Products Co. International lnc.. international Plaza.

Englewood Cliffs; New Jersey 07632) It) (im/l Protcose Peptone No. 3 (Difco) H) Gmll Tap water q.s.

Balance The presterilization pH is 7.0. The inoculated fermentation flasks are incubated at a temperature of 28 C. on a Gump rotary shaker operating at 250 r.'p.m. with a 2 /2 inch stroke. Ucon antifoam (a synthetic defoamer supplied by Union Carbide, N.Y., N.Y.) is used if needed. Harvest is usually after 5 to 12 days of fermentation. .v

The antibiotic titer of the fermentation beer can be monitored by an agar'plate'disc assay using the bacte-. rium Klebsiella pneumrmiae. This bacterium is inoculated'into the assay agar (Streptomycin Assay Agar,

BBL: Cockeysville, Maryland. 21030) of the following composition? Beef Extract l5 .Gmll

v Yeast Extract 3.0 Gni/l Gelysatc Pcptone. supplied by" Baltimore Biological Laboratories 6.0 (I'm/l Agar 15.0 Gm/l 'Dcionized water Balance adjust pH to 7.9 V

Sterilize'at 121 C. (-15 minutes.

Phosphate buffer 7 ent. The agar plates are incubated at 37} C. for l6- l 8 hours. Presence ofantibiotie U;44.590 is evidenced by the zone of inhibition around a paper diseto which a fermentation sample was previously applied. The diam eter of the zone of inhibition reflects the potency of the antibiotic sample. Thus, a mm. zone of inhibition using a 12.7 mm. paper disc to which 0.08 ml. of antibiotic samplehas been appliedis expressed as one bio unit per ml. (1 BU/ml.).

Part B. Recovery Whole fermentation beer (ca 1600 ml. assaying 5 BU/ml. against K. pneumoniae), obtained as described above; is filtered using diatomaceous earth as a filter aid. The filter cake is washed with water. The clear beer and'wash 1800 ml.) is then passed through an activated carbon column. The column measures 2.8 X 44 cm. and contains 126 grams of activated carbon. The

carbon column is washed with 1750 ml. water and the wash is discarded. The column is then washed with l litereach of of 1%, 2% and 5% acetonczwater concentration. These eluates are also discarded. The column is then eluted with 1 liter each of l0%, and 50% acetonezwater concentration. These eluates, which contain antibiotic U-44,590, are pooled and the acetone is removed on a rotatory evaporator at (2.115

No. steam pressure). 05.15

(0.1N pH 6.0) is used asthedilui usual run' are as follows:

A silica gel (Merck-Da'rmstadt Car. 7734) column is prepared from 420 grams of silica gel packed in methanol: chloroform lzl v/v.). The column measures 3.8 X

1 88 mm. Solid A, obtained as described above, is added on the top of the column and the column is then eluted with methanol: chloroform (l:l 'v/v). Active fractions,

I as determined by the above-described K. pneumoniae assay, are pooled and the solvent is removed from said I pooled fractions by use of a rotary evaporator at 30 C./l mm. Hg.; yield, 830 mg. assaying 7.5 BU/mg. of

antibiotic LI -44,590. I

Part C. PurificationNo. I i a A.preparation of. antibiotic U-4.4,590, obtained as de- "scribedabove in Part B., is subjected to chromatography on silica gel using the solvent system ethyl acetate:

is as follows:

A column of silica gel (Merck-Darmstadt, ll5 grams/gram of the U-44,590 preparation being chro-' matographed) methyl acetate: methanol'(6:l v/v) is prepared by pouring a slurry of silica in the solvent into the column to give a height-diameter ratio of 1.0:1 after 3 being packed. The U-44,59O preparation, obtained as I described above in Part B, is dissolved in methanol, silica is added (three times the weight of the U-44,590

preparation used), and this is then taken down to a dry powder on a rotatory evaporator at 40 mm. Hg. The resulting dry solid is added to the top of the silica column through a small head of the solvent ethyl aceta- .te:methanol (6:1 v/v). After a forerun of 4 liters, 50 ml.

fractions are collected and assayed for activity against K. pneumoniae. Active fractions are also tested for .methanol (6:1 v/v) to give a purer preparation containing U-44,590. The procedure for this purification step solids content. Fractions greater than 50 BU/mg are pooled and then taken to dryness in a rotary evaporator at 40 C./7 mm. Hg. to yield a-syrup. Fractions and their K. pn'eumoniae (K.p.) activity and solids from a Zone of e Inhibition wt. of solid Fraction i (using l2.7 in'Fraction Number mm. discs) (mgm) l l0 l6 34.5 I IS 30 I20 33 30.9 I25 35 i I 34 I 33 2l.7 I 32 I 3l 2L8 I 30 I 29 2L8 I 28 2s 17.9 2a j 200 27 l7.5 205 27 2 l() 26 14.4 2l5 26 220 26' I2.l 225 26 -Continued Zone of I Inhibition Wt. of solid Fraction (using 12.7 in Fraction Number mm. discs) (mgm) Fractions l20-l80, incl'. are pooled and [taken to dryness on a rotatory evaporator at 40/7 mm. Hg. to give a syrup, wt. 2.66g, assaying 54 K.p. BU/mg (Fractions 181-240, incl. give a syrup, 830 mg. 32 BU/mg., and fractions 241-300, incl. 'give a syrup, wt. 710 mg., as-

saying 11 BU/mg.) The standard assayed 4 BU/mg.

against the usual assay for this standard of 6 BU/mg. Part, D. Purification No. 2

- The preparations -of' U-44,59O obtained as described j in Part C., can be further purified to a preparation of essentially pure U-44,5 9 O by passage over anothersilica gel column using this time the solvent system methanol: methylene chloride (1:8 v/v). The procedure is as follows: i

A U-44,590 preparation, as obtained in Part C., (2,28 grams) is dissolved in methol and 7 grams of silica geL asdescribed in Part C., is added The solvent from this'rniitture is removed ona rotary evaporator at 40 Cl /7 The resulting solid is added to acolumn of silica get 750 g.,' 4.8 x 96cm, hold-upvolume 1500 mi, inadeup in mos-c3 01, (1:8 v/v)]. A forerun ('ll0 0 ml.) is collectedffollowed by 50 ml. fractioris. Fractions"l4l200, inclusive, weight 390 mg. when taken to dryness in the form of a syrup; This mat'erial shown to be almost pure U 44,590 by thin layer chromatography (tlc).

, V The tlc is conducted on silica gel plates using the solvent sys temMeOh-Ch Cl (1:9 v/v). Zones of the antibiotic are detected by spraying the plates with IOJM- nO spray, and with 50% aq. H 50 followed by heatrial in this solvent system is 0.1)1. I Part E. PurificationNo. 3 I

ing at j1=f10 c. for ca l0 min. The Rf of the active mate- Thepreparation ofantibiotic U-44,590 obtained in Part Dean. be further purified by acetylation of the preparation followed by deacetylation and crystallization.-The procedure for acetylation is as follows:

Aisample (ca. 22. g.) (WU-44,590 preparation, prepared as described in Part D and assaying I60 BU/mg,

is dissolved in pyridine (300 ml). and to this solution, stirred magnetically, is added acetic anhydride,(l50 ml ).over the'course of45 min. After standing overnight at room temperature, volatile materials are-removed as completely as possible on a rotatory evaporated at 40/l 5 mm. Hg., and finally under high vacuum. togive a tan syrup. g

This syrup is stirred with CH CI (200 ml), and a colorless, flocculent precipitate is removed by filtration and washed with CH CI until the washings are colorless. The precipitate is discarded. THe combined filtrate and washings are washed with' aqueous HCl (N/20, I00 ml) twice. the aqueous layer being acidic after the second wash. The-aqueous layers are discarded. The organic phase is then washed with water (100 ml). saturated aqueous NdHCO (10 ml). again with water 100 ml). and dried (NA SO The aqueous layers are discarded.

Removal'of solvent on a rotary evaporator at 40 and 15 mm. Hg. gives a dark syrup (21.10 g). which is dissolved in EtOAc (50 ml) by warming on a steam-bath. On cooling. crystallization occurs: the solid is removed by filtration. washed with EtOAc. and dried at 60.15 mm. Hg. to give essentially pure 3',5'-di-O-acetylated U-44.590 (12.01 g.. m.p. 123124.5 C.). Recrystallization from the same solvent gives 11-44590 diiacetate. having a melting point l24125 C. This compound is then labeled Ll-44.474. V

1.144.474 is deacetylated to afford essentially pure U-44.590 by the Zemplen procedure which is as follows.

The crystalline diacetate 1144.474 (24.90-g) is stirred magnetically in methanol (400 ml). and methanolic sodium mcthoxide (Stauffer Chem. Co. 257:. 5 drops) is added Stirring is continued till the solid has dissolved (Drierite tube). and the solution allowed to stand at room temperature for about 2 hours. Solid car bon dioxide. in small pieces. is then added cautiously. with stirring. to neutralize the methoxide. and the solvent is removed on a rotatory evaporator at 40 and mm. Mg.. giving a colorless oil.

The residue .is dissolved in methanol (50 ml) by warming on a st'eambath and diluted with ethyl acetate (50 ml). Crystallization occurs on cooling. The solid (12.39 g)-is collected on a sintered filter at the pump. washed with methanol. and dried in a vacuum oven at 60/'15 mm. Hg. Antibiotic U-44.590 crystallizes in colorles prismatic needles, mp. l41142. Removal'of solvent from the filtrate plus washings on the evaporator and crystallization from tiiethanol-ethyl acetate gives additional material (1.91 g. ni.p. l40.514l.5).

EXAMPLE 2 EXAMPLE 3 As disclosed in Example 2. various acylates of U- 44.59O can be made. and these 'acylatcs are useful to upgrade U-44.590. By following the procedure of Example I. Part E. the 3,5"-di-esters of U-44.590 are formed.

The 5-mono-esters can be formed by standard procedures using a minimum amount of acylating agent.

The 3-mono -esters and phosphate can be formed by tritylating U-44.590 to give the 5'-trityl derivative. ac-

"ylating this compound with the desired acylating agent.

selected from those disclosed above. to give the 3'- mo'no-ester 5'-trityl derivative. which then can be converted to the 3 mono-ester by removal 0 the trityl EXAMPLE 4 The 5-phosphate of U-44.590 can beprcpared by procedures as disclosed in the work of D. Mitsunobu.

K. Kato. and .l. Kimura [.l. Amer. Chem. Soc.. 91. 65 10 1969)]. This compound can be used for the same purposes as U-44.S90.

The compounds. described above. being the derivatives of U-44.590 which are within the scope ofthc subject invention. can be shown by the following structural formula N-CH wherein R and R are selected from the group. consisting ofa carboxylic acid acyl radical of from 2 to 18 carbon atoms inclusive; or a halonitro-. hydroxy-. amino-. cyano-. thiocyano-. and lower alkoxysubstituted hydrocarbon carboxylic acid acyl radical of from 2 to 18 carbon atoms, inclusive; R is hydrogen and R is as defined above or phosphate. or R is hydro' gen and R is a carboxylic acid acyl radical of from 2 to 18 carbon atoms. inclusive; or a halo-. nitro-. hydrox y-. amino-, cyano-. thiocyano-. and a lower alkoxysubstituted hydrocarbon carboxylic acid'aeyl radical of from 2 to 18 carbono atoms. inclusive. or phosphate.

Additional characterization data for U-44.474. pre pared as disclosed in Example 1. Part E. is as follows:

Elemental Analysis: Calcd. for C13H19N3O7 Found: C, 47.41; H, 5.82; N. 12.76; 0. 34.0].

Molecular Weight: 329 (Determined by mass spectrometry) Infrared Absorption Spectra: U-44,474 has a characteristic infrared absorption spectrum when suspended in a mineral oil mull. Peaks are observed at the following wave lengths expressed in reciprocal centimeters:

Band Frequency (Wave Numbers) Intensity mmtnwwwwmzg Band Frequency (Wave Numbers) Intensity I41 I 137) (oil) I358 I327 I318 I3I0 I298 I280 I250 I227 l I88 I I48 I I I3 I097 I057 I030 ll I I000 987 964 957 950 936 892 863 8224 79l 775 755 740 72] (oil) 675 668 U-44,474 also has a characteristic infrared absorption spectrum when pressed in a KBr disc. Peaks are observed at the following wave lengths expressed in reciprocal centimeters:

Band Frequency (Wave Numbers) Intensity -Conti'nued Band Frequency (Wave Numbers) Intensity l0l l We claim: 1. A process for preparing antibiotic U-44,59O having the structure which comprises cultivating Streptomyces platensis var. clurensis, having the identifying characteristics of NRRL 8035, in an aqueous nutrient medium under aerobic conditions until substantial antibiotic ,activity is imparted to said medium by the presence of antibiotic U-44,590.

2. A process, according to claim 1, wherein said aqueous nutrient medium contains a source of assimilable carbohydrate and assimilable nitrogen.

3. A process, according to claim 1, wherein said antibiotic U-44,59O is isolated from the fermentation broth.

UNITED STATES PATENT OFFICE QETTFICATE ()F CGRRECTTQN PATENT NO. 3,907, 645 DATED September 25, 1975 Page 1 of 2 'NVENTOR(5) 1 Clarence DeBoer and Brian Bann lster It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Col umn 1, l ine 19, for "According" read Accordingl y l ine 50, for 5440 M read 5400 M 5400 M Col umn 5, l ine 48, for "D iffo" read D i fco l ine 50, for 'Blooc" read Blood l ine 55, for"10 read 10' Column 4, l ine 51, for "recorder" read recorded l ine 44, for "A permanent" read A subcul ture of th is microorganism can be obta ined from the permanent Col umn 5, l ine 60, for "18.279-592]" read 18:279-592 Column 8, l ine 10, for

"as" read at Columns 15 and 14, at bottom of Table 5, Miss ing:

+ Good ut il izat ion Poor util ization Doubtful util ization No ut i l ization H H H ll Pridham, T.G., and D. Gottl ieb. 1948. The uti l ization of carbon compounds by some Actinomycetales as an aid for species determination. J. Bacteriol 56:107-114.

Column 15, line 47, for as assimilable" read an assimilable l ine 50, for "larctose" read lactose l ine 62, for "to steril i ization" read to steril ization Column 17,

l ine 52, For U-44,590 in read U-44,590 to give U-44,590 in Column 18, l ine 40, for "(15 No. read (15 l ine 65, for "each of of 1%" read each of a 1% l ine 65, for "each of 10%" read each of a 10% Col umn 19 l ine 7, for Car 7754)" read Cat 7754) l ine 55, For '40/15'mm. read 40 /15 mm. l ine 42, for "rota ry" read rotatory Col umn 20, l ine 42, for MeOh-" read MeOH- l ine 58, for "evaporated' read evaporator Column 21, l ine 2, For

. Page 2 of. 2 UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION PATENT NO. 3,907,6 5 DATED September 25, 1975 INVENTOR(S) Clarence DeBoer and Brian Bannister It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

"NdHCO read NaHCO l ine 5, for "(NA SO read (Na SO l ine 5, for "rotary" read rotatory l ine 9, for 60 .15 mm. read 60 /15 mm. l ines 54-35, For colerles" read colorless l lne 68, For "0" read of V Signed and Sealed this thirteenth Day of April 1976 [SEAL] A ttes t:

RUTH C. MASON C. MARSHALL DANN Arresting Officer (mnmissimu'r nj'Iatc/zls and wTrademurkx 

1. A PROCESS FOR PREPARING ANTIBIOTIC U-44,590 HAVING THE STRUCTURE
 2. A process, according to claim 1, wherein said aqueous nutrient medium contains a source of assimilable carbohydrate and assimilable nitrogen.
 3. A process, according to claim 1, wherein said antibiotic U-44,590 is isolated from the fermentation broth. 